Transcriptome-wide mapping of N3-methylcytidine modification at single-base resolution
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ABSTRACT:
PROVIDER: PRJNA1212065 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32037328_1.fastq.gz | Fastqsanger.gz | |||
| SRR32037328_2.fastq.gz | Fastqsanger.gz | |||
| SRR32037329_1.fastq.gz | Fastqsanger.gz | |||
| SRR32037329_2.fastq.gz | Fastqsanger.gz | |||
| SRR32037330_1.fastq.gz | Fastqsanger.gz |
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Project description:3-Methylcytidine (m3C), a prevalent modification of tRNAs, was recently identified in eukaryotic mRNAs. However, its precise distribution and formation mechanisms in mRNAs remain elusive. Here we develop a novel approach, m3C immunoprecipitation and sequencing (m3C-IP-seq), utilizing antibody enrichment to profile the m3C methylome at single-nucleotide resolution. m3C-IP-seq captures 12 m3C modification sites in cytoplasmic tRNA isoacceptors and 2 in mitochondrial tRNA isoacceptors. Moreover, m3C-IP-seq permits the comprehensive profiling of m3C sites in mRNAs and lncRNAs, with their presence reliant on a nuclear isoform of METTL8. A significant proportion of m3C sites is concentrated in the 3’ untranslated region (3’ UTR) of mRNAs. Additionally, m3C methylation is dynamic and responds to hypoxia. Functionally, m3C formation in the 3’ UTR is associated with mRNA degradation. Collectively, our data demonstrate the widespread presence of m3C modification in the human transcriptome and provide a resource for functional studies of m3C-mediated RNA metabolism.
2025-03-19 | GSE287299 | GEO
Project description:3-Methylcytidine (m3C), a prevalent modification of transfer RNAs (tRNAs), was recently identified in eukaryotic messenger RNAs (mRNAs). However, its precise distribution and formation mechanisms in mRNAs remain elusive. Here, we develop a novel approach, m3C immunoprecipitation and sequencing (m3C-IP-seq), utilizing antibody enrichment to profile the m3C methylome at single-nucleotide resolution. m3C-IP-seq captures 12 cytoplasmic tRNA isoacceptors and 2 mitochondrial tRNA isoacceptors containing m3C modifications. Moreover, m3C-IP-seq permits the comprehensive profiling of m3C sites in mRNAs and long noncoding RNAs, with their presence reliant on a nuclear isoform of METTL8. A significant proportion of m3C sites is concentrated in the 3' untranslated region (3' UTR) of mRNAs and is associated with mRNA degradation. Additionally, m3C methylation is dynamic and responds to hypoxia. Collectively, our data demonstrate the widespread presence of m3C modification in the human transcriptome and provide a resource for functional studies of m3C-mediated RNA metabolism.
| S-EPMC11897884 | biostudies-literature
Project description:Deoxyuridine (dU) in DNA can result from deamination of cytosine and dUMP misincorporation. The easy single-nucleotide resolution assay for dU are crucial to understanding its role in genome. Herein, we present a new concept of “base substitution” for accurate single-nucleotide resolution profiling of dU. The method termed AI-Seq (Artificial Incorporation of a modified nucleobase for sequencing) was developed using artificial cytosine (N3-C) to replace dU. After the artificial base construction, dU can read as cytosine during polymerase chain reaction assay. AI-seq was validated on synthetic DNA and then applied to the genome of HEK293T cell line. Collectively, the “base substitution” provides a novel approach for generating comprehensive information about the distribution of dU and can be potentially adapted to detect other epigenetic modifications.
2021-03-22 | GSE142071 | GEO
Project description:DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays critical roles in a variety of biological and pathological processes in mammals. In active DNA demethylation, the ten-eleven translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution are still highly desirable. Herein, we propose a Chemical Labeling Enrichment and Deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by A3A and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mouse embryonic stem cells. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.
2023-08-31 | GSE239943 | GEO
Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel nTET-assisted deaminase sequencing (TAD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied TAD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. TAD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
2024-06-30 | GSE254203 | GEO
Project description:5-Methylcytosine (5mC) is a crucial epigenetic modification plays a significant role in the regulation of gene expression. Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes. In this study, we develop a novel NaegleriaTET-assisted deaminase sequencing (NTD-seq) method for the base-resolution and quantitative detection of 5mC in genomic DNA. The TAD-seq method utilizes a Naegleria TET-like dioxygenase (nTET) to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A), creating an A3A mutant (A3Am). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged. Consequently, TAD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites. We applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
2025-04-02 | GSE263426 | GEO
Project description:We established N3-Kethoxal molecule to probe RNA secondary structure in vivo at transcriptome level.
2020-02-22 | GSE122096 | GEO
Project description:Protocol for detecting m5C RNA modification at single-base resolution using m5C-TAC-seq
| PRJNA1166655 | ENA
Project description:In this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine their DNA methylation level at single-base resolution in a cost-effective way. In addition, we have developed a new alternative method, MRB-seq (MeDIP-repetitive elements removal-bisulfite sequencing), which interrogates 5mCs in functional regions by depleting nearly half of repeat fragments enriched by MeDIP. Comparing MB-seq and MRB-seq to whole-genome BS-seq using the same batch of DNA from YH peripheral blood mononuclear cells. We found that the sequencing data of MB-seq and MRB-seq almost reaches saturation after generating 7-8 Gbp data, whereas BS-seq requires about 100 Gbp data to achieve the same effect. In comparison to MeDIP-seq and BS-seq, MB-seq offers several key advantages, including single-base resolution, discriminating the methylated sites within a CpG and non-CpG pattern and overcoming the false positive of MeDIP-seq due to the non-specific binding of 5-methylcytidine antibody to genomic fragments.
2017-08-17 | GSE94071 | GEO
Project description:Active DNA demethylation in mammals involves TET-mediated iterative oxidation of 5-methylcytosine (5mC)/5-hydroxymethylcytosine (5hmC) and subsequent excision repair of highly oxidized cytosine bases 5-formylcytosine (5fC)/5-carboxylcytosine (5caC) by Thymine DNA glycosylase (TDG). However, quantitative and high-resolution analysis of active DNA demethylation activity remains challenging. Here we describe M.SssI methylase-assisted bisulfite sequencing (MAB-seq), a method that directly maps 5fC/5caC at single-base resolution. Genome-wide MAB-seq allows systematic identification of 5fC/5caC in Tdg-depleted embryonic stem cells, thereby generating a base-resolution map of active DNA demethylome. A comparison of 5fC/5caC and 5hmC distribution maps indicates that catalytic processivity of TET enzymes correlates with local chromatin accessibility. MAB-seq also reveals strong strand asymmetry of active demethylation within palindromic CpGs. Integrating MAB-seq with other base-resolution mapping methods enables quantitative measurement of cytosine modification states at key transitioning steps of active demethylation pathway, and reveals a regulatory role of 5fC/5caC excision repair in active DNA demethylation cascade. Analysis of 5fC/5caC excision repair-dependent active DNA demethylome by MAB-seq in mouse embryonic stem cells.
2014-11-02 | E-GEOD-62631 | biostudies-arrayexpress